Selective and non-invasive visualization or treatment of vasculature

ABSTRACT

Disclosed are methods and materials for visualizing or treating vasculature. The methods and materials in this invention relate to selective and non-invasive visualization, or treatment such as chemical occlusion, of vasculature in the mammalian eye. The methods utilize fluorescent dyes and tissue-reactive substances encapsulated within heat-sensitive liposomes which are subsequently heated to release the contents thereof at a pre-determined anatomical locus. The methods of this invention further utilize tissue-reactive agents which, when activated, are effective to cause localized tissue damage and occlusion of blood vessels and/or blood sinuses. The materials of this invention relate to diagnostic reagents and kits for visualizing or treating a mammalian blood vessel or sinus.

GOVERNMENT SUPPORT

Work described herein was supported in whole or in part by Grant No. EY07768, awarded by The National Institutes of Health. The Government hascertain rights in the invention.

CONTINUING APPLICATION INFORMATION

This application is a divisional application of U.S. application Ser.No. 08/355,619, filed Dec. 14, 1994, and now issued as U.S. Pat. No.5,935,942.

FIELD OF THE INVENTION

The present invention relates generally to methods and materials forvisualization or treatment of vasculature.

BACKGROUND OF THE INVENTION

Of the estimated 34 million people in the United States who will be age65 or older in 1995, approximately 1.7 million will have some visualimpairment resulting from age-related macular degeneration (ARMD).Approximately 100,000 of those affected will experience a devastating,rapid loss of vision due to choroidal neovascularization (CNV). (See,for example, U.S. Dept. Health and Human Serv., (1994) National AdvisoryEye Council (1990-1992).) ARMD is the most common cause of vision lossin people over age 50 and, as the population ages, a greater number ofelderly persons will become blind from ARMD than from glaucoma anddiabetic retinopathy combined. Leibowitz et al. (1980) 24 Surg.Ophthalmol. (suppl.) 335-610; Sorsby (1972) in: Ministry of HealthReports on Public Health and Medical Subjects (128th ed., London);Ferris (1983) 11 Am. J. Epidemiol. 132-151.

Clinical research has shown that laser treatment of CNV reduces the riskof extensive scarring in selected cases of CNV characterized by awell-defined, predictable fluorescein angiographic pattern covering anarea limited in size. Unfortunately, such "classic cases" comprise only25% of the population with CNV, leaving 75% of the patients at risk ofbecoming blind from macular disease without the benefit of lasertreatment. Moreover, the frequent (54%) recurrence of CNV is mostlyattributed to incomplete angiographic visualization and subsequentinadequate treatment of CNV. Macular Photocoagulation Study Group (1986)104 Arch. Opthalmol. 503-512. The practical difficulty in detecting CNVclinically has been documented in a recent study involving clinical andpathological examinations of 30 eyes. In 57% of the cases, CNV wasdetected histologically but not clinically. Sarks (1973) 75 Br. J.Ophthalmol. 587-594. This finding is in agreement with otherhistopathological studies in which CNV was detected inangiographically-unrecognized lesions. Bressler et al. (1992) 110 Arch.Ophthalmol. 827-832; Small et al. (1976) 94 Arch. Ophthalmol. 601-607.

As presently performed, the ability of fluorescein angiography tohighlight CNV is limited by a number of factors. First, the dye rapidlyfills both the retinal and choroidal vessels. Thus, visualization ofsmall vascular beds, such as those typical of CNV, is often hampered bythe lack of contrast caused by the bright fluorescence emanating frommajor choroidal vessels. Second, visualization of CNV is based on theleakage into and/or staining of tissue by the dye, which occurs onlyduring a particular pathological stage. This process is not reliablebecause at certain stages of the disease, diseased vessels do not leakor stain. Also, metabolic waste products may accumulate in the vicinityof the lesion, decreasing the permeability or delaying the leakage intoextravascular tissues. Third, when vessels leak, dye accumulates in thetissues surrounding the CNV lesion and actually masks its boundaries.Fourth, both the exciting and fluorescent light may be absorbed bysubretinal blood, turbid fluid, pigment, or fibrous tissue, therebyreducing the intensity of the fluorescence emanating from the CNV.Bresler et al. (1988) 32 Surg. Ophthalmol. 375-413; Bressler et al.(1991) 109 Arch. Ophthalmol. 1242-1257.

Indocyanine green (ICG) angiography has been reportedly beneficial insome cases. Destro et al. 96 Ophthalmology 846-853. Because theexcitation and emission wavelengths of this particular dye are longerthan those of fluorescein, the light penetrates turbid media better,thereby eliminating the fourth limitation mentioned above. On the otherhand, however, the enhanced penetration of light in ICG angiographyaggravates the first-mentioned limitation of fluorescein angiography,i.e., interfering fluorescence, because large underlying choroidalvessels are visualized more effectively. Moreover, ICG angiographyshares with fluorescein angiography the limitation of relying on leakageand staining of extravascular tissues. The poor understanding of thestaining and pooling mechanisms of this dye hampers interpretation ofangiograms.

The lack of adequate methods of angiographic visualization isunfortunate because clinical research has shown that laser treatment canreduce, in the long term, the risk of extensive loss of vision inclassic CNV. (See, for example, Bressler et al. (1991) 109 Arch.Ophthalmol. 1242-1257.) In addition, the failure of laserphotocoagulation has been attributed to inadequate identification of theentire extent of CNV and its location relative to the fovea.

As mentioned, CNV is commonly treated by laser photocoagulation in whicha thermal scar is produced. The procedure typically causes a dramaticloss of vision when the fovea is treated. See, for example, MacularPhotocoagulation Study Group (1991) 109 Arch. Ophthalmol. 1220-1231.Nonetheless, the treatment is performed to prevent progressive visualloss. The cases eligible for treatment, which make up only 25% of theeyes with CNV, must be the well-defined "classic" type of CNV that isnot too large. The remaining 75% of affected eyes are untreated becauselaser photocoagulation does not spare useful vision. In addition, newblood vessels recur in a majority of the patients (54%) treated withlaser photocoagulation, thereby necessitating further scarringtreatment. Other than the incomplete identification of CNV mentionedabove, recurrence has been attributed to damage to Bruch's membrane andscarring, conditions known to predispose tissues to new blood vesselgrowth.

It is an object of this invention to provide methods and materials forselective occlusion of vasculature. That is, it is an object of thisinvention to provide for the occlusion of blood vessels withoutsignificant, concomitant damage to tissue surrounding and supplied bysaid blood vessels. It is another object of the instant invention toprovide methods and materials for selective and non-invasive chemicalocclusion of blood vessels and sinuses in the mammalian eye, especiallyblood vessels and sinuses of choriodal origin. It is a further object ofthe instant invention to provide methods for selectively andnon-invasively occluding vascular abnormalities of the mammalian eye,such abnormalities being associated with macular degeneration andrelated clinical conditions involving neovascularization, such aschoroidal neovascularization. It is still a further object of theinstant invention to provide methods for non-invasively occludingvascular abnormalities associated with pathologies of thechoriocapillaris such as choroideremia, gyrate atrophy, and acuteplacoid multifocal pigment epitheliopathy, as well as vascularabnormalities which are non-choroidal such as those associated withdiabetes. It is yet another object of the instant invention to providediagnostic reagents and diagnostic kits for selective, non-invasivechemical occlusion of vasculature. These and other objects and featuresof the invention will be apparent from the description, drawings andclaims which follow.

SUMMARY OF THE INVENTION

The present invention provides a method of chemically occluding a bloodvessel or blood sinus in a mammalian eye which involves:co-administering intravenously a fluorescent dye encapsulated withinheat-sensitive liposomes and a tissue-reactive agent which is effectiveto cause chemical tissue damage following its activation; non-invasivelyheating tissue at a pre-determined anatomical locus within the eye sothat the heat-sensitive liposomes leak and release their contents intothe blood vessel or sinus at the pre-determined locus; exciting thefluorescent dye; visually observing a pattern of fluorescent vasculaturewhich develops at the pre-determined locus; and, activating thetissue-reactive agent disposed within the blood vessel or sinus so thatthe blood vessel or sinus is chemically damaged to an extent sufficientto occlude the vessel or sinus. As used herein, the steps of the methodof the instant invention are collectively referred to as "laser-targetedocclusion". In some cases, the steps which precede activation of thetissue-reactive agent are collectively referred to as "laser-targetedangiography", "laser-targeted delivery," or "laser-targetedvisualization." The present invention contemplates that the steps of themethod of laser-targeted occlusion can be repeated for an amount of timethat the liposomes are circulating systemically following theiradministration. Similarly, the present invention contemplates that thesteps of laser-targeted angiography can be repeated for an amount oftime that the liposomes are circulating systemically following theiradministration.

In another embodiment of the method of the instant invention, the methodof chemically occluding a blood vessel or sinus in a mammalian eyeinvolves: co-administering intravenously a fluorescent dye and atissue-specific factor co-encapsulated within heat-sensitive liposomes,said tissue-specific factor being effective to impair growth orregeneration of vasculature; non-invasively heating tissue at apre-determined anatomical locus within the eye so that theheat-sensitive liposomes leak and release their contents into the bloodvessel or sinus at the pre-determined anatomical locus; exciting thefluorescent dye; visually observing a pattern of fluorescent vasculaturewhich develops at the pre-determined anatomical locus; and, exposing theblood vessel or sinus at the locus to the tissue-specific factordisposed within the blood vessel or sinus so that the growth orregeneration of the blood vessel or sinus is impaired.

In yet another embodiment, the instant invention provides a method ofoccluding vasculature in a mammalian eye which involves: administeringintravenously heat-sensitive liposomes having a fluorescent dyeencapsulated therein; irradiating a pre-determined anatomical locuswithin the eye with a first laser beam to selectively and non-invasivelyheat the vasculature at the locus so that the liposomes accumulated atthe locus release their contents into the vasculature at the locus;identifying a blood flow origin within the vasculature by visualizing anadvancing blood/dye boundary within a feeder blood vessel that suppliesblood to a vascular abnormality at the locus; and, occluding the feederblood vessel with a second laser beam focused on the blood/dye boundary.

In another aspect, the invention features materials for use in themethod, e.g., a diagnostic reagent and a diagnostic kit each comprisinga fluorescent dye encapsulated within heat-sensitive liposomes and atissue-reactive agent. Alternatively, the diagnostic reagent and kiteach comprise a tissue-reactive agent co-encapsulated with fluorescentdye within heat-sensitive liposomes. In yet another embodiment, theinstant invention contemplates that the diagnostic reagent and kit eachcomprise a fluorescent dye which is a tissue-reactive agent. Thediagnostic kit of the instant invention optionally further comprisesmeans for encapsulating fluorescent dye, either alone or together withtissue-reactive reagent, within heat-sensitive liposomes.

The method of laser-targeted angiography and occlusion of the instantinvention solves problems encountered in conventional fluorescein or ICGangiography. For example, the local selective release of a fluorescentdye according to the method of the instant invention permitspre-determined vascular beds to be visualized without interference fromfluorescence emanating from overlying or underlying beds. Second,visualization according to the instant invention is independent ofstaining and leakage of dye into tissue surrounding a region of abnormalvasculature, such as choroidal neovascularization (CNV). Rather, itrelies solely on the presence of a dye in the vascular lumen. Thus, theCNV can be visualized as long as it is patent, i.e., open to blood flow.This feature also simplifies interpretation of angiograms. Third, theshort time during which release of the encapsulated dye occurs,accompanied by rapid and eventual clearance thereof, ensures that thedye does not accumulate outside the vessels, and thus does not mask theCNV as do conventional materials and methods. Fourth, the hemodynamicsof the CNV, delineated by the progress of the dye, using the methods ofthe instant invention, allow the vessels feeding the CNV to beidentified. Such identification allows the clinician to selectivelyocclude an identified feeder vessel. In this manner, large areas ofretina overlaying the CNV could be spared, thus limiting the unnecessaryvisual loss typically associated with prior art methods ofphotocoagulation. Fifth, angiograms performed in accordance with theinstant invention can be repeated for at least about 45 minutes as longas the liposomes are circulating in the blood. This providesopportunities to correct errors in alignment of optical equipment and toperform angiography of both eyes during the course of a single patientvisit and/or single therapeutic procedure.

All the above advantages can be demonstrated using the methods andmaterials of the instant invention which permit successful visualizationof classic and occult CNV, as well as visualization of thechoriocapillaris, even in the presence of scar tissue. Laser-targetedangiography as described herein detects and delineates CNV and CNV-typelesions effectively. This increases the number of patients who couldbenefit from such therapy, reduce the recurrence rate now attributed tolack of adequate visualization, and restrict the treated area, therebyreducing the amount of visual loss accompanying occlusion treatment.

As disclosed herein, a method of occluding CNV that does not cause ascar and spares the overlying retina and adjacent choriocapillaris ishighly desirable and can be met by the instant invention's methods oflaser-targeted occlusion. In one embodiment, the method of the instantinvention combines laser targeted delivery and photodynamic therapy bytargeting delivery of a photosensitive agent-to the CNV and occludingthe CNV by photosensitization of the agent. Such a method solves many ofthe limitations of systemic photodynamic therapy and those ofconventional thermal laser photocoagulation. Thus, the tissue-reactiveagent can be released specifically in the choroid, thus avoiding releasein the retinal vessels. Similarly, the agent also can be released withinthe CNV while avoiding release in the retina. Next, by irradiating onlyafter the agent's release into the pre-determined anatomical locus,tissue damage can be limited essentially to the vessels that areselectively perfused by the agent. The lack of accumulation of the agentin the interstitial tissues prevents their subsequent damage uponactivation. Third, as disclosed herein, there are clear indications thatCNV's are perfused by a slower blood flow than the normalchoriocapillaris. Therefore, the agent could be released and the tissueirradiated only after enough time has elapsed to ensure clearance fromthe normal choriocapillaris. This sequence would preclude damage to thechoriocapillaris which is crucial to the maintenance of the retinalpigment epithelium. Fourth, laser-targeted occlusion of CNV according tothe instant invention allows both "classic" and "occult" type lesions tobe amenable to treatment, as the skilled practitioner now canselectively occlude the CNV while sparing the retina and thus preservingvision. Fifth, laser-targeted occlusion does not cause a scar as doesthermal occlusion, thereby reducing risk of recurrence ofneovascularization.

The foregoing and other objects, features and advantages of the presentinvention will be made more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and features of this invention, as wellas the invention itself, may be more fully understood from the followingdescription, when read together with the accompanying drawings, inwhich:

The FIGURE is a diagrammatic representation of the instrumentation usedto perform laser-targeted angiography and occlusion.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As will be described below in greater detail, the instant inventionrelates to methods and materials for occluding vasculature.Specifically, the methods and materials of the instant invention relateto selective and non-invasive chemical occlusion of vasculature in themammalian eye. The methods utilize fluorescent dyes and tissue-reactivesubstances encapsulated within heat-sensitive liposomes. These liposomesare subsequently heated to release the contents thereof at apre-determined anatomical locus. The methods of this invention furtherutilize tissue-reactive agents which, when activated, are effective tocause localized tissue damage and occlusion of blood vessels and/orblood sinuses. The materials of this invention relate to diagnosticreagents and kits for identifying and chemically occluding a mammalianblood vessel or sinus.

As used herein, the term "occluding" means obstructing blood flow in ablood vessel or sinus. The term "occlusion" means a physical blockage orimpediment in a blood vessel or sinus that obstructs passage of bloodtherethrough. An occlusion can develop immediately as the direct resultof treatment, or it can gradually develop as a consequence of an earliertreatment. Occlusion can occur when there is an impenetrable blockage inan existing vessel or sinus, or as a result of treatment whichsubsequently impairs the growth or regeneration of a blood vessel orsinus. Occlusion of vasculature can be caused chemically or thermally,as desired. As used herein, an occlusion may be either permanent ortemporary.

The term "chemically occluding" means any non-thermal means ofoccluding. Chemical occlusion is the result of treatment of vasculaturewith a chemical, biological, pharmaceutical, or pharmacological agentwhich causes physiological and/or structural damage to vasculature. Forexample, "chemical damage" means damage caused by toxic radicals,cross-linking radicals or the equivalents thereof; damage caused bybiological moieties which bind, interact with, or otherwise affectvascular integrity and/or modulate vascular growth or regeneration; or,damage caused by pharmaceutical or pharmacological compounds such as avasoconstrictor or a vasodilator. "Tissue damage" refers to damage toblood vessel or blood sinus tissue originating within the lumen, such asoriginating within the endothelial cell lining of the lumen, and whichsubsequently progresses into an occlusion. Tissue damage can be causedchemically as described above or thermally such as with a laser beam."Interstitial tissue" refers to non-vascular tissue, e.g., retinal orsubretinal tissues surrounding and supplied with nutrients by bloodvessels.

As used herein, the term "administration" or "co-administration" isintended to mean any form of delivery into a recipient's blood supply,such as by intravenous injection or infusion. The only modes of deliveryexcluded from use in the instant invention are those which damage thecapsular integrity of liposomes and/or cause the liposomes' contents tobe released prematurely, i.e., prior to the intentional release of theircontents by heating as described herein.

As used herein, the term "fluorescent dye" is intended to mean anyfluorescent substance suitable for use in the instant invention. As willbe understood by the skilled practitioner, fluorescent substancessuitable for use in the instant invention include those which aresuitable for administration to mammals, including humans. It will befurther understood that fluorescent substances amenable toencapsulation, or which can be rendered amenable to encapsulation, aresuitable for use in the instant invention. Moreover, substances whosefluorescence is quenched when in an encapsulated form at an appropriateconcentration as described herein also are suitable for use in themethods and materials of the instant invention. Thus, dyes such as6-carboxy-fluorescein and similar fluorescein derivatives are suitablefor use herein. Particularly useful fluorescent substances are thosewhich can be excited by and emit light at wavelengths not stronglyabsorbed by opaque tissue lesions, blood and/or biological pigments.Such substances readily can be detected in eye tissue comprisinglesions, blood or biological pigments. In this regard, indocyanine green(ICG) also is suitable because its excitation is not strongly absorbedby opacities, blood, or biological pigments. Similarly, aluminumphthalocyanine tetrasulfonate (AlPcS₄) is an exemplary fluorescentsubstance and tissue-reactive agent for the reasons discussed hereinbelow, however, other equivalent substances would also be suitable.Identification of equivalents is well within the skill of the ordinarypractitioner and would require no more than routine experimentation.

As used herein, the term "tissue-reactive agent" is intended to mean anagent competent to react adversely with vascular tissue such that thevascular tissue is chemically damaged. Tissue-reactive agents includethose competent to generate upon activation toxic radicals orcross-linking substances which physiologically and/or structurallydamage vascular tissue. For example, tissue-reactive agents canpreferentially interact with tissue components disposed on the innerlumen walls of mammalian vasculaturel interaction with these lumencomponents results in an adverse effect on the associated vasculaturewhich progresses into an occlusion. Additionally, tissue-reactive agentscan be photosensitive agents activated by light to produce tissuedamage. An example of one such photosensitive agent useful in the methodof the present invention is aluminum phthalocyanine tetrasulfonate."Tissue-reactive by-products" refer to the activation by-products oftissue-reactive agents which may form within the vasculature followingactivation and act chemically to damage the vascular tissue.

As used herein, the term "heat-sensitive liposomes" includes liposomesformulated using physiologically compatible constituents, such asdipalmitoylphosphatidyl-choline and dipalmitoylphosphatidyl-glycerolphospholipids, that permit preparation of liposomes using art-recognizedtechniques that release their contents at temperatures above those ofthe mammalian body temperature, i.e., above 37° C. Upon exposure totemperatures at least about 4° C. above mammalian body temperature,"release" occurs by leakage or seepage of the liposomes' contents or byactual lysis (complete or incomplete) of the liposomes. The instantinvention contemplates use of heat-sensitive liposomes which arenegatively-charged unilamellar liposomes less than about 450 nm indiameter to facilitate handling, administration, unhindered progressthrough mammalian vasculature, and minimize side effects, e.g.,interference with the mammalian blood clotting cascade.

The method of the instant invention features, in certain embodiments,heat-sensitive liposomes which contain a quenching concentration offluorescent dye. The term "quenching concentration" refers to thatconcentration of dye which is sufficiently high to mask or minimizedetection of fluorescence even-when illuminated with a suitable lightsource. The physics of fluorescence quenching are well-known in the artand the skilled artisan would be able to determine a quenchingconcentration for any fluorescent dye through no more than routineexperimentation. When the heat-sensitive liposomes used in the methodsof the instant invention are heated, the contents of the liposomes leakinto and/or are released into the lumen of the blood vessels in whichthey have accumulated, thereby causing a dilution in the concentrationof the fluorescent dye by the plasma within the vessels which results ina non-quenching concentration of dye, e.g., a concentration that visiblyfluoresces upon sufficient excitation with a suitable light source.

As used herein, the term "non-invasively heating" means heating withoutcausing substantial damage to tissue, including scarring orphysiological impairment. Non-invasive heating as accomplished by theinstant invention typically involves irradiating tissue with a laserbeam at a pre-determined depth by manipulating wavelength and pulseduration. Methods of the instant invention contemplate a laser fornon-invasively heating which has a wavelength absorbed by blood andocular pigments. According to a preferred embodiment of the instantinvention, a blood vessel or sinus is selectively and non-invasivelyheated to a temperature of approximately 41° C. by irradiating with alaser beam having a wavelength absorbed by blood, for example, 488 nm.Such non-invasive heating by irradiating tissue with a suitable laserbeam pre-disposes the heat-sensitive liposomes of the instant inventionto release their contents into the vasculature at the tissue locuswithout causing substantial damage to the vascular and extravascular(interstitial) tissue at that locus or elsewhere, while minimizingheating of extravascular tissue elsewhere.

As used herein, the phrase "pattern of fluorescent vasculature" meansthe hemodynamic distribution of fluorescence which develops at apre-determined anatomical locus immediately following release andexcitation of the fluorescent dye at the locus. The vasculature subjectto selective heating displays a distribution of fluorescence whichdelineates the topology of the vasculature at the locus. The displaychanges as the fluorescence advances downstream or upon the end of therelease, as fresh blood not carrying released fluorescence flows intothe locus. A pattern as contemplated by the instant invention includesnot only this time-dependent feature, but also includesvasculature-dependent features. An exemplary vasculature-dependentfeature involves the differential distribution of fluorescence in normalblood vessels versus abnormal blood vessels. Fluorescence will clearfrom blood vessels with normal blood flow earlier than from those bloodvessels with subnormal blood flow. Thus fluorescence persists in vesselswith sub-normal blood flow. Such abnormal blood vessels, therefore,display a delayed clearance of the released dye. Patterns of fluorescentvasculature developed through practice of the methods described hereincan therefore be used to identify a vascular abnormality and/orimplement occlusion of such an abnormality. For example, the pattern offluorescence which develops according to the instant methods may be apattern of vasculature originating from the choroid. An abnormal bloodvessel or sinus with persistent fluorescence may be an abnormal choroidblood vessel or sinus associated with choroidal neovascularization.Another important feature of the pattern of the instant invention isthat it is relatively visually unimpaired by fluorescence emanating fromoverlying or underlying vascular beds. This is so because the method ofthe instant invention permits selective release of fluorescent dye at apre-determined anatomical locus, thereby avoiding confoundingfluorescence in adjacent vascular beds. In the case of a patternemanating from the choroid, interference by vessels of the retina andthe large choroidal vessels is substantially eliminated by the methodsof the instant invention.

The methods of the instant invention are suitable for occluding avascular abnormality. A "vascular abnormality" refers to, for example, aphysiologically atypical vascular feature, including structural as wellhemodynamic abnormalities. A vascular abnormality may be the consequenceof a congenital or developmental irregularity, an injury, a disease, aneoplasm, or aging. Often, it is associated with vision impairment orloss. Macular degeneration is a vascular abnormality particularlysuitable for occlusion by the methods and materials of the instantinvention. In particular, choroidal neovascularization (CNV), includingclassic and occult choroidal neovascularization, can be occluded usingthe methods and material of the instant invention. Additionally, avascular abnormality refers to a pathology of the choriocapillaris suchas choroideremia, gyrate atrophy, acute placoid multifocal pigmentepitheliopathy, and the like. Moreover, the methods and materials of theinstant invention are suitable for occlusion of vascular abnormalitiesassociated with other disease such as diabetes which affectnon-choroidal vasculature.

As used herein, the term "activating" or "activation" is intended tomean the process by which the tissue-reactive agent is induced orstimulated to effect chemical damage and occlusion of vasculature. Theexact manner in which an activated tissue-reactive agent effectuatesthis chemical damage and occlusion depends on the type oftissue-reactive agent used. For example, a tissue-reactive agent may bechemically inert or non-reactive until it is exposed to an activatingprocess such as irradiation with a suitable laser beam. In oneembodiment of the instant invention, the tissue-reactive agent isactivated by irradiating the blood vessel or sinus at a pre-determinedanatomical locus with a laser beam, the wavelength of which beingsufficient to activate the agent. A wavelength suitable for use in theinstant invention is one which sufficiently activates thetissue-reactive agent and does not itself cause substantial damage totissue at the pre-determined anatomical locus. For example, whenaluminum phthalocyanine tertrasulfonate is used as the tissue-reactiveagent in the method of the instant invention to occlude CNV-typevasculature, irradiation of the vasculature with a laser wavelength ofapproximately 675 nm is sufficient to cause activation of the agent. Thephotoactivated agent then causes chemical tissue damage which results inocclusion. Thus, the term activation contemplates any process by whichan agent is selectively stimulated or induced to manifest certainproperties which lead to occlusion as that term is defined herein. Inaccordance with the methods of the instant invention, activationpreferably is accomplished after clearance of the fluorescent dye andthe agent from vasculature with normal blood flow, and before clearanceof the dye and the agent from vasculature with subnormal blood flow.Alternatively, activation and release of the agent may occursimultaneously rather than sequentially. In this particular embodiment,activation is preferably accomplished before observable leakage of thedye or agent into tissue adjacent a particular blood vessel or sinus, orbefore significant observable clearance of the dye or agent from thesite of their release from the heated liposomes. The terms "normal bloodflow," "sub-normal blood flow," and "clearance" as used herein aredefined below.

"Normal blood flow" refers to the movement of blood through mammalianvasculature which is unimpeded and progressing underphysiologically-normal pressures and at physiologically-normal rates.Normal blood flow may be found in blood vessels having mature diameters."Sub-normal blood flow" refers to the movement of blood throughmammalian vasculature which is not progressing at physiologically-normalrates, and perhaps also not progressing under physiologically-normalpressures. Sub-normal blood flow may result from a stricture in thevasculature caused by disease, or may be associated with damage ordisease in adjacent vasculature. Sub-normal blood flow may also be theresult of blood vessels having immature diameters, tortuous paths orexcessive length, such as newly formed blood vessels. "Clearance" refersto the disappearance of, or diminishment of, an agent released in theblood vessels and/or vasculature at a particular anatomical locus.Typically, clearance is associated with displacement, by fresh fillingblood, of the blood into which the agent had been released earlier. Forexample, clearance is observed upon the diminishment of the fluorescenceemitted by the fluorescent dye.

In a currently preferred embodiment of the method of the instantinvention, the method of selective and non-invasive laser-targetedocclusion involves: administering intravenously heat-sensitive liposomeshaving encapsulated within a tissue-reactive agent and a fluorescentdye; irradiating a pre-determined anatomical locus within the eye with afirst laser beam so that vasculature at the pre-determined locus isselectively and non-invasively heated and the heat-sensitive liposomesaccumulated at the locus leak and release their contents into thevasculature at the locus; exciting the released fluorescent dye;monitoring clearance of the fluorescent dye from blood vessels withnormal blood flow while simultaneously monitoring persistence of the dyewithin blood vessels having sub-normal blood flow so that a vascularabnormality can be visualized at the pre-determined anatomical locus;and, irradiating with a second laser beam the blood vessels withsubnormal blood flow by focusing the second laser on vessels containingpersistent fluorescent dye so that the tissue-reactive agent alsopersisting in such vessels is activated, resulting in chemical damage toand occlusion of the blood vessels with subnormal blood flow. As used inthis preferred embodiment, "first laser beam" refers to a laser beamhaving a wavelength absorbed by blood and ocular pigments. Additionally,a first laser beam contemplates one which selectively heats tissue,including its blood vessels or sinuses, to a temperature ofapproximately 41° C. without causing substantial physiological damage tothat tissue or vasculature. As used in this preferred embodiment, "asecond laser beam" refers to a laser beam which is suitable to activatethe tissue-reactive agent without causing substantial physiologicaldamage to the vasculature in which it is disposed. Further, the secondactivating laser beam causes minimal damage to adjacent extravascular(interstitial) tissue.

As used herein, the terms "persistence" and "clearance" are relativeterms intended to mean lingering, detectable fluorescence versusfluorescence which disappears and/or diminishes (in a time-dependentmanner, e.g., synchronized with the cardiac cycle) to the extent thatits detection quickly becomes negligible. Normal blood flow may beassociated with vasculature which exhibits early clearance whilesubnormal blood flow may be associated with vasculature which exhibitslingering, persistent fluorescence and delayed clearance. As discussedabove, the methods of the instant invention effectively exploit thisfluorescent differential to identify normal and non-normal vasculature.Thus, the step of the instant invention relating to activating thetissue-reactive agent preferably is accomplished after clearance of thedye and the agent from vessels with normal blood flow, and beforeclearance from vessels with subnormal blood flow.

In accordance with the embodiments discussed above, the method of theinstant invention involves encapsulating substances in artificial lipidvesicles called liposomes, injecting them intravenously, and releasingtheir contents in the selected tissue by non-invasively heating theblood vessels therein. Using these embodiments of the instant method,for example, dyes and tissue-reactive agents can be released into aspecific portion of the subretinal choroidal vasculature to enablevisualization of the choriocapillaris even in the presence of subretinallesions, without relying on leakage or staining.

To enhance the specificity of release of the dye and tissue-reactiveagent into the choroidal vasculature, for example, the heat must beconcentrated maximally in the region of interest. Heat or laser-energyabsorbing substances in this region are blood, waste products,occasionally extravasated blood, the retinal pigment epithelium (RPE)and the choriocapillaris. In the case of a choroidal disease such asCNV, the CNV is about 20 μm thick and the choriocapillaris is of asimilar thickness. By matching the heating laser wavelength to anabsorption peak of blood or ocular pigments, most of the energy isdeposited in the region of interest. At about 488 nm, the RPE absorbsthe fist 40 to 75% of the light, and the CNV absorbs 25% of theremaining light. Thus, even without taking into account the absorptionof the waste products, 55 to 81% of the energy is absorbed. The presenceof a minimal amount of extravasated blood (50 μm thick) is sufficient toleave an insignificant amount of energy beyond the RPE. In areas void ofCNV, for example, the RPE and the choriocapillaris absorbs 55% to 81% ofthe energy, thus also letting little light penetrate into the choroid.In other words, most of the energy is deposited within 20 μm of the RPE.

Upon heating, the temperature distribution inside a retinal regionoverlaying an area of RPE heated by approximately 4° C. can becalculated from well-established first principles. For a heated disc 800μm in diameter, the rise in retinal temperature is about 2.8° C. at adistance of 150 μm, i.e., where the first retinal capillaries arelocated. This indicates that the temperature rise in the tissuesurrounding the retinal vessels is not sufficient to cause heating ofliposomes and release therefrom in retinal capillaries. Retinal vessels(typically 50 μm or less in diameter in the central macula) absorb somelight but this only causes a heat rise of less than about 0.5° C.

Thus a CNV and its vicinity will reach the releasing temperature of theliposomes while the retinal tissues will not. Consequently, releasingsubstances such as dyes and tissue-reactive agents in the choroid, butnot releasing in the retina can be accomplished using the methods andmaterials of the instant invention.

In accordance with the instant invention, the fluorescent dye releasedinto a CNV and the choriocapillaris, for example, yield a laser-targetedangiogram permitting visualization of the vasculature at apre-determined anatomical locus. During the early phases of the release,released substances fill the vessels at the locus. Fluorescence andtissue-reactive agents subsequently clear from the choriocapillaris inapproximately 120-240 msec. As disclosed herein, clearance fromvasculature associated with CNV or a CNV-type lesion is delayed. This isbased, in part, on the fact that the path from the level of thechoriocapillaris to the CNV and back to the choriocapillaris is longerthan the 270 μmeter diameter of a choroidal lobule within the underlyingand/or surrounding choriocapillaris. The circuit of flow of blood in theCNV is in parallel with that of the circuit of flow in thechoriocapillaris. Thus the delayed clearing of vasculature associatedwith a CNV-type lesion permits hilighting of the lesion after thefluorescence has cleared from the underlying and surroundingchoriocapillaris when the methods and materials of the instant inventionare utilized. In essence, this means that fluorescence will remain for alonger period of time within the CNV-type lesion, thereby permitting thepractitioner to precisely delineate and localize the site of the CNVlesion and occlude the vasculature, for example, by activating thetissue-reactive agent also persisting at the site.

One preferred embodiment of the present method relies on instrumentationsuch that release of fluorescent dye and tissue-reactive/photosensitiveagent from the heat-sensitive liposomes is accomplished with an argonlaser at 488 nm and activation of the tissue-reactive agent isaccomplished with a laser diode at 675 nm. Use of two separate lasersallows delay of activation after the agent's release and only until theagent has cleared from the choriocapillaris. This enhances the damage tothe CNV while minimizing the effect on the choriocapillaris. In anotherembodiment, release and activation of the tissue-reactive agent can beaccomplished simultaneously with the same laser beam. To titrate thepower of the laser and ensure that the agent is being released, theinstant invention contemplates fluorescent dye in liposomes injectedsimultaneously with the agent; laser-targeted angiography according tothe present invention is then used to visualize the agent's release. Theactual anatomical area covered by the activating beam is tailored insize and depth to the anatomical area of the CNV perfused by the agent.

As illustrated in the Figure, the light for activation of thetissue-reactive agent originates from a diode laser operating atapproximately 675 nm. The laser output is focused into a fiber 16 by anoptical coupler 15. The output of the fiber is collimated by a lens 17and diverted toward a fundus camera 31 by a dichroic mirror 10reflecting in red. A second mirror 18 then directs the beam into theimaging path of the camera. The desired size of the beam is obtained byselecting the numerical aperture of the fiber and the focal length ofthe collimating lens. The power and the duration of the activating pulsewill be controlled from a computer via the analog input port of thediode laser console. In addition, a hard-wired shutter 14 with a fixeddelay will ensure limited exposure should the computer malfunction. Thepower of the activation beam is also monitored by a photosensor 21. Amore detailed description of the instrumentation suitable for use in theinstant invention, and its application in the methods of the instantinvention, follows below in Example 1.

In operation, laser-targeted angiography in accordance with the presentinvention may be performed first, without use of a second laser. Thepreferred image acquisition and processing routines allow immediatefeedback on the location of the CNV. The time necessary to allowclearance of the dye from normal vessels can be assessed, firstmanually, and then, numerically from the angiograms. The subsequentsteps then consist of releasing the contents of the liposomes andturning the excitation laser on after the pre-determined delay.

Yet another embodiment of the method described herein further comprisesco-administering a tissue-specific factor encapsulated withinheat-sensitive liposomes which is effective to impair growth orregeneration of a blood vessel or blood sinus. In a preferredembodiment, the instant invention provides a method of chemicallyoccluding a blood vessel or blood sinus including the step ofco-administering intravenously a fluorescent dye and a tissue-specificfactor, both of which being encapsulated within heat-sensitiveliposomes. The term "tissue-specific factor" means any moiety whichmanifests an affinity for vascular tissue and/or its individualcomponents and affects said tissue adversely, such as by causing tissuedamage, inhibiting tissue growth or inducing tissue regression invasculature. Moreover, it is further contemplated that a tissue-specificfactor is one which binds to endothelial cell receptors of a growing orregenerating blood vessel or blood sinus. In the instant invention, atissue-specific factor may be an antibody such as an antibody directedagainst vascular endothelial growth factor or fibroblast growth factor.Impairment of growth or regeneration refers to a disruption in thenormal growth or reparative processes usually exhibited by vasculartissue; the disruption may be permanent or temporary.

Additionally, the laser-targeted occlusion method of the instantinvention may further comprise co-administering an anti-inflammatoryagent or an antibiotic encapsulated within heat-sensitive liposomes.Antibiotic is understood to include anti-bacterial, anti-fungal,anti-neoplastic and anti-parasitic antibiotics. Anti-neoplasticantibiotics include, but are not limited to, the aclacinomycins,bleomycins, chromomycins, mitomycins, and the olivomycins (see the MerckIndex, 11th edition, 1989).

In yet another embodiment, the instant invention provides a method ofoccluding vasculature in the mammalian eye involving the steps of:administering intravenously heat-sensitive liposomes having encapsulatedwithin a fluorescent dye; irradiating a pre-determined anatomical locuswithin the eye with a first laser beam in order to selectively andnon-invasively heat the vasculature and cause the liposomes accumulatedtherein to release their contents; identifying abnormal blood vessels orsinuses and a blood flow origin within abnormal vasculature byvisualizing an advancing blood/dye boundary within the feeder vesselsupplying blood to the vascular abnormality; and, occluding the feederblood vessel with a second laser beam focused on the blood/dye boundary.The term "blood/dye" boundary describes an interface, i.e., adiscernible point of distinction, between non-fluorescent filling blood,i.e., freshly entering blood devoid of released dye, and draining blood,i.e., fluorescent blood into which dye and/or tissue-reactive agents hadbeen earlier released. The blood/dye boundary need not be sharplydelineated to be successfully identified and localized. It is enough forpractice of the methods described herein that this boundary signifies arelative separation of filling blood and draining blood. A "feeder bloodvessel" refers to a vessel or complex of vessels which supplies blood toa particular system of vasculature at a particular anatomical locus. Afeeder blood vessel may supply a normal vascular system with blood, oralternatively it may supply an abnormal vascular system with blood. Thisparticular embodiment of the invention contemplates at least twodifferent modes of occlusion using a second laser beam. The first modeinvolves a second heating laser beam which is effective to cause heatdamage to the feeder blood vessel such that the vessel is coagulated andoccluded. "Heat damage" means thermally-induced damage such as by laserburning. "Coagulated" or "coagulation" refers to the physiologicalcondition of a heat-damaged vessel following laser burning. In thisfirst mode, occlusion is the result of deliberate heat damage, but theheat damage is confined to the blood/dye boundary. In contrast, a secondmode of occlusion contemplates a tissue-reactive agent which, uponirradiation with a second activating laser beam focused on saidblood/dye boundary, causes chemical damage to the vasculature tissue soit becomes occluded; this particular mode does not cause heat damage orcoagulation.

In another aspect, the instant invention further provides a diagnosticreagent for identifying and chemically occluding a blood vessel or bloodsinus in a mammal. The diagnostic reagent comprises a fluorescent dyeencapsulated within heat-sensitive liposomes; a tissue-reactive reagenteffective upon activation to cause chemical damage to tissue; and, apharmaceutically acceptable vehicle. "Pharmaceutically acceptablevehicle" refers to any vehicle which is suitable for systemicadministration to a mammal, particularly administration into the bloodstream. Thus, for example, a suitable vehicle is a physiologicallybalanced, aqueous salt solution. In another embodiment, thetissue-reactive agent is encapsulated within heat-sensitive liposomes,either alone or together with the fluorescent dye. In yet anotherembodiment, the fluorescent dye also is the tissue-reactive agent.

The instant invention also provides a diagnostic kit for identifying andchemically occluding a blood vessel or blood sinus in a mammal. Thediagnostic kit of the instant invention comprises a fluorescent dye; atissue-reactive agent effective upon activation to cause chemical damageto tissue; and, means for encapsulating said dye within heat-sensitiveliposomes. In another embodiment, the kit comprises an additional meansfor encapsulating the tissue-reactive agent within heat-sensitiveliposomes, either alone or together with the fluorescent dye. In yetanother embodiment, the tissue-reactive agent and the fluorescent dyeare the same. As used herein, the term "means for encapsulating" refersto any means suitable for encapsulating the above-described componentswithin heat-sensitive liposomes as defined herein. The means may be indesiccated form, dehydrated form, or otherwise in non-liquid form. Ifnon-liquid means are provided, the diagnostic kit of the instantinvention optionally further comprises a suitable rehydrating reagent.Alternatively, the means may be in liquid form, ready-to-use, or mayrequire pre-mixing. Preparing liposomes such as those used in theinstant invention is well-known in the art and the skilled artisan wouldbe able to devise such encapsulating means through no more than routineexperimentation.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the invention in any way.

EXAMPLE 1 Instrumentation, Image Acquisition, and Image ProcessingEXAMPLE 1.1 Delivery of Liposome Release Beam

A suitable delivery means consists of an argon laser for the release.

The releasing beam is delivered through the illumination arm of a funduscamera (Zeiss). In this camera, the illumination path is designed todeliver an annulus of light at the pupil plane leaving the centerunexposed to light and reserved for the imaging path. When the pupil islarge the illumination path is unobstructed but, if the pupil ispartially dilated, or if a small animal is examined, the iris blockspart of the illumination. Clipping by the iris hinders the constant andpredictable delivery of power through the releasing beam. To overcomethis limitation, the releasing beam is delivered via the imaging path.This scheme facilitates alignment through the pupil and enhancesreliability. The reliability stems from the fact that, by viewing theimage on the fundus and optimizing it, one centers the imaging path inthe pupil.

The coupling of an argon laser 32 to the fundus camera 31 is achieved,as shown in the Figure, at the conjugate image plane originally used forthe eyepiece. The argon laser 32 is fed into a fiber by a laser to fibercoupler 1. The fiber is one of the arms of a fused fiber optic coupler 2which channels part of the power toward colliminating lens 8. The fiberoutput is collimated, projected on the conjugate plane of the retina anddirected into the imaging path by a dichroic filter 11 reflecting blue(488 nm). The focal length of the collimating lens 8 and the numericalaperture of the fiber are chosen to yield the desired beam spot on theretina. The dichroic filter 11 maximizes the amount of light directedtoward the eye while minimizing the reflected light reaching acharge-coupled-device (CCD) camera 33. The reflected blue light reachingthe CCD camera 33 is further reduced by green band pass filter 12 usedalso in fluorescein angiography to block the excitation and pass thefluorescence. The image of the fundus is demagnified and focused on theCCD camera 33 with the aid of lens assembly 13.

To visualize the beam during alignment, an infrared light source 6 isadded coaxially to the fiber via the fused fiber optic coupler 2. Theimage of the beam exiting the delivery assembly 34 is visualized on amonitor. The beam is directed to different regions on the fundus bymoving the location of delivery assembly 34 on the conjugate plane. Theposition of this assembly is adjusted by a motorized translation stage35 attached to the body of fundus camera 31 and controlled via computerby a joystick.

The power of the laser is measured at a conjugate plane of the pupil bythe light partially reflected by a thin glass 19 toward a focusing lens20 and a photosensor 21. The size of the mirror matches the diameter ofthe aperture to restrict the measurement to the part of the beam that isdirected into the eye.

The laser pulse for liposome release is controlled by a computercontrolled shutter 9. A hard-wired fixed-delay shutter 22 preventsaccidental long exposures in case of computer malfunction.

EXAMPLE 1.2 Delivery of Illumination Beam

The blue light illumination necessary for angiographic visualization ispresently delivered through a fiber assembly replacing the originalflash unit. This arrangement is adequate for large pupils but can causelosses in smaller animals with pupils that are narrower than the annulusof light.

The illumination beam originates from the second arm of the fibercoupler splitter 2 fed by the argon laser 32. The pulse duration isdetermined by the laser internal shutter under the control of thecomputer. The output of the fiber illuminates, via a collimating lens 3,the conjugate image plane of the retina and is placed at a planeconjugate to the pupil. By forming a small spot at the pupil the beameases the alignment into the pupil.

EXAMPLE 1.3 Operation of the System

The operation of the system is synchronized through a computer with theaid of a program written with virtual instrumentation software. Thecamera signal is used for the synchronization. Upon activation by a footpedal, the video recorder is started, the location on tape is recordedin a file, the argon laser 32 is turned on, the releasing beam deliveryshutter 22 is opened for a given period and, after a preset delay, theillumination is turned off followed by the video recorder.

EXAMPLE 1.4 Image Acquisition

The images first are acquired on a Betacam video recorder. The videorecorder can be replaced by a frame grabber capable of recording 24Mbytes of images. This allows spanning of a sequence of 3 seconds at arate of 16 images per second. After each delivery, the data can becompressed and stored on an optical drive.

Unless the images are digitized on line, they can be retrieved foranalysis from the video tape. The playback of the video recorder iscontrolled by the computer. The digital locations on tape, recordedduring the experiment, permit selection of a given sequence and digitizeit in parts with the current frame grabber equipped with 4 Mbytes ofmemory.

EXAMPLE 1.5 Image Processing

The most basic routine consists of subtracting the angiogram immediatelypreceding the release from the sequence following the liposomes release.This permits isolation of the dynamic changes. There is little motionduring a sequence and no need for registration.

Once the static background has been subtracted, a number of imageenhancement algorithms can be applied. As mentioned above, flow in theCNV is slower than that in the normal tissue. Thus, it is feasible todifferentiate between the clearance rate in the normal choriocapillarisfrom that in the CNV. Windows of interest, placed in different regionsof the image, are used to plot the intensity versus time and measure thedecay rate. A distribution curve of the decay rates in the differentwindows can thereby be generated. Since the clearance of thechoriocapillaris is relatively homogeneous in the posterior pole, onecan obtain a distribution of clearance rates with a sharp peak at a highvalue, corresponding to the normal areas, well separated from thedistribution of lower rates due to the CNV. Experience permits theidentification of a reliable cutoff to isolate the CNV clearance rates.This then yields a time in the sequence after which the remainingfluorescence is only in sluggish vessels, namely in the CNV. The framesfrom this time on are then combined to yield a map of the CNV. Thereliability of this or alternative algorithms can be verified bycomparisons with histology.

EXAMPLE 2 Reagent Preparation

Liposomes were prepared in accordance with standard techniques using twolipids, DPPC and DPPG (Avanti Polar Lipids, Pelhan, Ala.) withoutfurther purification to generate liposomes with a phase transition of41° C. Examples relating to the use of liposomes for delivery ofsubstances in the mammalian body are disclosed in U.S. Pat. No.4,310,506; U.S. Pat. No. 4,350,676; U.S. Pat. No. 4,515,736; U.S. Pat.No. 4,522,803; U.S. Pat. No. 4,610,868; U.S. Pat. No. 4,891,043; andU.S. Pat. No. 5,257,970, the disclosures of which are hereinincorporated by reference. Large unilamellar vesicles were obtained byreverse-phase evaporation using art-recognized techniques such as thosedescribed by Magin et al. (1984) 34 Chem. Phys. Lipids 245-256, orforced extrusion following freeze-thaw cycles as described by Hope etal. (1985) 812 Biochem. Biophys. Acta 55-65. The mean diameter of theliposomes was measured by light scattering (Nicomp, Goleta, Calif.).Liposomes occupied only a fraction of the volume in the preparation.

6-Carboxyfluorescein (Molecular Probes, Junction City, Ore.) waspurified on a hydrophobic column and diluted to approximately 100 mMusing art-recognized techniques such as those described in Zeimer et al.(1988) 29 Invest. Opthalmol. Vis. Sci. 1179-1183. A sufficient dose forbaboons weighing 9 to 12 kg is approximately 14 mg/kg; typically theconcentration was 13.3 mM. This corresponds to a 1.5 ml/kg dose ofliposome suspension injected intravenously. The concentration of the dyeencapsulated in the liposomes was tested by measuring the fluorescenceof the dialyzed preparation before and after lysis with detergent. Themeasured concentration is compatible with the release of the original100 mM concentration if the liposomes occupy one seventh of the volume.This concentration is well within the range of concentrations that causefluorescence quenching. On release in the plasma, the dye was dilutedand fluoresced strongly.

As will be understood by the skilled practitioner, fluorescentsubstances suitable for use in the instant invention include those whichare suitable for administration to mammals, including humans. It will befurther understood that fluorescent substances amenable toencapsulation, or which can be rendered amenable to encapsulation aresuitable for use in the instant invention. Moreover, substances whosefluorescence is quenched when in an encapsulated form at an appropriateconcentration as described herein also are suitable for use in themethods and materials of the instant invention. Thus, dyes such as6-carboxyfluorescein and similar fluorescein derivatives are suitablefor use herein. Particularly useful fluorescent substances are thosewhich can be excited by and can emit light at wavelengths not stronglyabsorbed by opaque tissue lesions, blood and/or biological pigments.Such fluorescent substances readily can be detected in vivo in thepresence of biological opacities. In this regard, indocyanine green(ICG) also is suitable because its excitation is not strongly absorbedby opacities, blood, or ocular pigments. Similarly, aluminumphthalocyanine tetrasulfonate (AlPcS₄) is an exemplary fluorescentsubstance and tissue-reactive agent for the reasons discussed hereinbelow, however, other equivalent substances would also be suitable.Identification of equivalents is well within the skill of the ordinarypractitioner and would require no more than routine experimentation.

Similarly, aluminum phthalocyanine tetrasulfonate (AlPcS₄) is presentedas an exemplary fluorescent dye and tissue-reactive agent for thereasons discussed below, however, other equivalent dyes would also besuitable for use in the instant invention. Again, identification ofequivalents is well within the skill of the ordinary practitioner andwould require no more than routine experimentation.

Aluminum phthalocyanine tetrasulfonate (AlPcS₄) is well-known in the art(see, for example, U.S. Pat. No. 5,166,197, the disclosure of which isherein incorporated by reference) and is considered useful as atissue-reactive agent because its peak absorption around 675 nm assurespenetration through blood (only 11% absorption by a 100 micron layer).Further, it is water soluble and can be encapsulated efficiently. It hasone of the highest known absorption coefficients (30 times higher thanthat of hematoporphyrin derivatives) ensuring high sensitization withminimal amount of light on the retina. It is a well-defined compoundwhich can be synthesized with high purity. Finally, it is removed within24 hours from the blood thereby reducing the period of light sensitivitywhich has been a significant limitation of other photosensitive agents.This tissue-reactive, photosensitive agent has been demonstrated to benon-toxic in a number of species and no side effects have been reportedwhen it was used intraperitonially in a few patients.

Aluminum phthalocyanine tetrasulfonate (Porphyrin Products, Logan, Utah)can be stored at 20° C. in the dark in the powder form. It is dissolvedin sterile water for injection and filtered through a 0.2 micron syringefilter. Liposomes of the instant invention are prepared followingmethods well-known in the art such as those described in detailpreviously by Zeimer et al. (1988) 29 Invest. Ophthalmol. Vis. Sci.1179-1183.; Zeimer et al. 30 Invest. Ophthalmol. Vis. Sci. 660-667; and,Hope et al. (1985) 812 Biochem. Biophys Acta 55-65.Dipalmitoylphosphatidyl-choline (DPPC) anddipalmitoylphosphatidyl-glycerol (DPPG) were obtained from Avanti PolarLipids (Pelham, Ala.) and used without further purification.

Raw materials were tested to ensure their sterility and glassware wasautoclaved. Liposomes were prepared using standard methods and aseptictechniques and tested for sterility by incubating them at 37° C. onblood agar plates and in thioglycollate media for 72 hours usingstandard materials and methods. The art-recognized limulus amebocytelysate (LAL) test was used to ensure that the liposomes and water wereessentially free from pyrogens, especially endotoxins. Since the sizedistribution of the liposomes affects their half-life in the bloodstream and since particles greater than 5 microns could cause clotting,the liposome preparation was filtered through a 0.4 micron polycarbonatefilter using standard materials and methods. The integrity of theliposomes was monitored by verifying that the concentration of theunencapsulated substance increased significantly after exposure ofliposomes to detergent.

EXAMPLE 3 Regulation of the Primate Retinal Microcirculation

Understanding the regulation of the retinal microcirculation isimportant, because there is evidence that, in most vascular diseases,the earliest pathology occurs at the capillary level. The capillary bedsin the retina next to the fovea are organized in two basic layers; adeep one, in the inner nuclear layer, and a more superficial one, in theganglion cell and nerve fiber layer. A more detailed examination hasrevealed a further subdivision into four layers closely matching thethickness of different neuronal layers and indicating a link betweenmetabolic demand and the arrangement and density of themicrovasculature. The multilayer arrangement leads to a plexus ofnumerous capillaries fed by a single relatively large arteriole.Consequently, the retinal microvasculature is subjected to a largepressure head, and changes in its resistance are likely to play asignificant role in blood flow regulation.

Laser-targeted angiographic methods of the instant invention wereapplied to study the response of the macular circulation tophysiological challenges. The local metabolic demand was increased byflickering of light, a stimulus which has been shown to induce bloodflow changes. The illumination was chopped and within 10 secondsfollowing the end of the flicker, a sequence of three laser-targetedangiograms was obtained. Three baboons were used in the study and themeasurements were performed on two occasions in each animal. Thearterial transit time, the capillary transit time and the fluorescenceintensity of each capillary bad were measured. In response to flicker,the blood flow in retinal arteries increased by 30%. The response of themicrocirculation was not homogeneous. It showed a maximum increase inthe mid perifoveal region where there is an increase in ganglion cellsand nerve fibers. The maximum change in the index representing capillaryblood flow exceeded (p<0.08) the change in the blood flow in the artery.It appears that, in response to an increased metabolic demand, aredistribution of blood occurs in the retina whereby more blood isdirected to the inner (superficial) capillary layer which is providedwith an apparently lower resistance due to its architecture. Thisregulation would be beneficial in preserving the supply to the innertissues, which consist of neuronal tissue remote from the choroid.

The application of laser targeted angiography as performed using methodsof the instant invention permitted, for the first time, the study of thelocal regulation of the retinal microcirculation. The results indicatethat the regulation is intimately coupled to the specific demand of thetissue it perfuses and thus varies across the macula and the depth ofthe retina.

EXAMPLE 4 Visualization and Hemodynamics of the Non-Human PrimateChoriocapillaris

Limited information is available on the physiology and pathophysiologyof the choroidal microvasculature, mainly due to the difficulty invisualizing it in vivo. Conventional fluorescein and even indocyaninegreen angiography are limited by the fluorescence emitted from the largechoroidal vessels which dominate the angiographic image and hindervisualization of the faint fluorescence from the thinnerchoriocapillaris. While a technique based on the subtraction ofconsecutive dynamic angiograms has minimized this large background(Flower (1993) 34 Invest. Ophthalmol. Vis. Sci. 2720-2729),visualization still depends on an ill-defined feature relating to therelative dynamics of vascular beds. The fact that slowly perfused onesmay not be imaged is a significant limitation. In addition, conventionalintravenous injection of dyes yields an ill-defined dye front, must belimited to few boli, and cannot be synchronized with the cardiac cycle.

Using the materials and methods of the instant invention, laser-targetedangiography for visualizing the choroidal microvasculature in non-humanprimates has been accomplished. The local release of dye from theheat-sensitive liposomes in the choroidal arteries was carrieddownstream and generated a dye front which reached the choriocapillaris.An intense and predominant fluorescence from the choroid was observed inthe absence of significant retinal fluorescence. The contribution of thechoriocapillaris to the image was dominant compared to that of the deepchoroidal vessels. Of particular notice was the lack of released dye inthe retinal vasculature and the dominant contribution of thechoriocapillaris to the angiographic image compared to that of the deepchoroidal vessels. The dynamic angiograms obtained in accordance withthe methods of the instant invention revealed the filling and emptyingof the choriocapillaris lobules. The dye was first cleared out at thecenter of polygon-shaped areas and disappeared radially to form ahypofluorescent zone surrounded by a polygonal outline. The filling andclearing pattern of the choroidal microcirculation was unchanged duringthe different phases of the cardiac cycle. This pattern of filling andclearing resembles the one described by Hayreh (1974) Graefes Arch.Clin. Exp. Ophthalmol. 165-179, and substantiates the concept ofchoroidal lobules each supplied at its center by an arteriole anddrained by annular venous channels as described by Torcyzynski et al.(1976) 81 J. Ophthalmol. 428-440 and Yoneya (1987) 105 Arch. Ophthalmol.681-687. Since anatomically the choriocapillaris forms a continuous bed,the segmental pattern revealed by the laser-targeted angiography methodof the instant invention must be attributed to the dynamics of thecirculation. This pattern can be explained by the presence of a pressuregradient between the central arteriole and the surrounding venules whichforces flow from the arterioles into the venules and prevents flow intothe adjacent network of capillaries. The average size of the lobules(n=76) was 270+90 μm in diameter. Adjacent arteries typically suppliedneighboring clusters which fit together like a jig-saw puzzle. A feedingartery often supplied remote islands of the choriocapillaris.Alternatively, clusters were observed with unfilled areas. The minimallateral diffusion of the dye indicates that there is no dynamiccommunication between adjacent clusters under physiologic conditions.

As further evidence of the extent of the present invention's successfulvisualization, a choroidoretinal scar was created by delivering a highpower laser pulse over a large (one disc diameter) area of the posteriorpole with the ND:YAG laser. A conventional fluorescein angiogram wasdominated by hyperfluorescence at the margin of the lesion and byhypofluorescence at the center of the scar. The angiogram did notprovide any information on the underlying choroidal vasculature. Incontrast, the laser-targeted angiograms of the instant inventionrevealed perfused clusters of choriocapillaris both in the region of thehyperfluorescence and at the center of the scar. The results demonstratethat thin nets of subretinal vessels, such as the choriocapillaris, canbe visualized in non-human primates by the laser-targeted angiographymethod of the instant invention while conventional fluoresceinangiography does not provide any clinically-useful information.

EXAMPLE 5 Visualization of CNV

A rat model of a CNV-type lesion was created by heavy laser burnsfollowing a well-established protocol such as the one described by Dobiet al. (1989) 107 Arch. Oohthalmol. 264-269. Although the choroidalnature of the newly-formed vessels resulting from such a protocol hasbeen previously documented, histology was performed to confirm that thenew vessels were located above Bruch's membrane as in human CNV. Tohighlight patent vessels, the histological method of Kues et al. wasused as described in 13 Bioelectro. 379-393 (1992). This method involvesinfusing the animal with horseradish peroxidase (HRP) prior tosacrifice, embedding the appropriate section of the eye in plastic usingstandard techniques and subsequently reacting with diaminobenzidine theHRP in the 2 micron histologic sections using art-recognizedhistological protocols. This yields an orange staining in patentvessels, i.e., those open to blood flow. Histology of the periphery of aburn demonstrates that the new, CNV-type vessels are close to thechoriocapillaris, thus resembling human CNV. Retinal pigment epithelian(RPE) cells were seen covering the CNV. The choroidal nature of thesevessels was further confirmed in another section of the same lesion byobserving their connection to the choroid through the Bruch's membrane.In this embodiment of the instant invention, the photosensitive agentwas released locally by a laser pulse and also activated to cause therelease of free radicals.

The CNV-type lesions were then studied with conventional fluoresceinangiography and the laser-targeted angiography method of the instantinvention. In the case of "classic" CNV, so defined by a well-delineatedleakage of dye on conventional fluorescein angiography, laser-targetedangiography yielded far superior visualization of the CNV-type lesion.Using laser-targeted angiography, the dye highlighted the normalchoriocapillaris as well as the abnormal pattern of CNV vessels. Thenormal choriocapillaris cleared rapidly while the CNV revealed asluggish flow. These results provide experimental proof that, at leastin this model, CNV has an abnormal slow flow. The sluggish flow alsocaused less dilution of the dye and thus yielded a bright fluorescence.The rapid clearance of the normal choroid had the dramatic effect ofleaving the CNV clearly delineated. In addition to delineation of theCNV, laser-targeted angiography revealed the dynamics of the flow in theCNV. The origin of the flow, namely localization of the local feedingvessels, could be identified clearly and the drainage of the dye intothe surrounding normal choroid provided evidence that these pathologicalvessels were of choroidal origin thus confirming their classification asa CNV.

Finally, the data indicated that multiple liposome releases--even in aCNV shown to leak on conventional fluorescein angiography--do not causeaccumulation of dye such that the visualization is impaired. Fluoresceinangiography performed at 14 seconds and 2 minutes after injectionrevealed the presence of a lesion with progressive leakage which istypical of "classic" CNV. A lack of sharp delineation of the CNV wasobserved. Live video images indicated that the leakage was localizedbelow the neurosensory retina. In contrast, laser-targeted angiographyrevealed a CNV with an exact location and flow pattern. Angiogramsobtained 17, 165 and 560 msec after the end of the dye release,respectively, depicted a brightly fluorescent abnormal pattern ofvessels and diffusely fluorescent patches. The abnormal pattern of thebrightly fluorescent vessels is typical of a CNV. The diffuse patcheswhich evolved rapidly correspond to the choriocapillaris. The samelobular pattern was observed in regions devoid of lesions. An angiogramobtained at 2 seconds showed that the fluorescent dye cleared from thechoriocapillaris while it remained in the CNV, indicating a sluggishflow. The advance of the dye clearly delineated the direction of flow inthe CNV. Subsequent releases in other regions of the CNV revealed asimilar pattern of flow thus demonstrating its anatomical nature.Additionally, although the angiograms were obtained after seven previousreleases performed within five minutes, there was no significantaccumulation of dye and no deterioration in visualization. Thepre-release image was subtracted from the subsequent images to highlightthe dynamic changes.

In a case of "occult" CNV, so defined by the lack of leakage or by anill-defined area of leakage on conventional fluorescein angiogram,laser-targeted angiography clearly delineated a CNV and its flow patternin a manner similar to that of the above-described "classic" case.Fluorescein angiograms obtained at 29 seconds and 78 seconds afterinjection revealed the presence of patchy fluorescence which did notevolve with time providing no indication of the presence of a CNV. Incontrast, laser targeted angiography revealed a CNV with its exactlocation and flow pattern. Angiograms obtained 50, 110 and 430 msecafter the end of the dye release, respectively, depicted a brightlyfluorescent abnormal pattern of vessels and fluorescent patches. Theabnormal pattern of the brightly fluorescent vessels is typical of aCNV. The patches evolved rapidly into a lobular pattern characteristicof choriocapillaris. Laser-targeted angiography at 1.2 seconds showedthat the fluorescent bolus cleared from the choriocapillaris while itremained in the CNV, indicating a sluggish flow. Subsequent releases inother regions of the CNV revealed a similar pattern of flow thusdemonstrating its anatomical nature.

Thus, this application of laser-targeted angiography to release dye inthe choroid yielded unique angiograms of the choriocapillaris anddemonstrated that laser-targeted angiography is a powerful tool forphysiological studies of this vascular tissue. Laser-targetedangiography is a tool of choice to study the patency or flow of thechoriocapillaris in diseases which may well be due to early pathologiesof the choriocapillaris such as choroideremia, gyrate atrophy, and acuteplacoid multifocal pigment epitheliopathy.

The most significant implication of these results is that laser-targetedangiography in accordance with the instant invention is useful for thediagnosis of ARMD-related CNV. Laser-targeted delivery suppliesinformation currently unavailable. The CNV can be delineated in"classic" and "occult" cases alike, thus demonstrating thatlaser-targeted angiography is not dependent, like conventionalangiography, on leakage from the CNV. In addition, the identification ofthe origin of the flow allows treatment of feeder vessels.

EXAMPLE 6 Targeted Occlusion of Vessels EXAMPLE 6.1 Laser-TargetedOcclusion In Iris Vessels: Rodent

Laser-targeted occlusion depends on the discovery disclosed herein thata tissue-reactive or photosensitive agent may be activated during itsvery short presence in the vessel lumen after its release from theheat-sensitive liposomes. In this example, a photosensitive agent,aluminum phthalocyanine tetrasulfonate (AlPcS₄,) was used because itspeak absorption around 680 nm assures penetration of fluorescencethrough blood (only 11% absorption by a 100 gm layer). This and otheradvantages of AlPcS₄ over other photosensitizing agents have beendiscussed above. Photosensitive agents such as AlPcS₄ are well-known inthe art (see, for example, U.S. Pat. No. 4,889,129 and U.S. Pat. No.5,166,197, the disclosures of which are herein incorporated byreference).

To demonstrate that the method of laser-targeted occlusion of theinstant invention can occlude vessels, a customary model which yieldsart-recognized and unequivocal documentation of its efficacy was used.The iris vessels of the albino rat are considered the model of choicebecause they are readily visible and untreated portions could be used aswell-defined and stable controls. It is within the skill of the ordinarypractioner to modify the experimental conditions described herein toextend the methods of the instant invention to a pigmented eye. Theprinciples exemplified by the rat model are applicable to non-humanprimate and human subjects. The protocol now described was designed inaccordance with the ARVO Resolution on the Use of Animals in Research.The experiments were conducted on six albino male Sprague Dawley ratsweighing 250 to 300 gm. They were anesthetized with ketamine (50 mg/kg)and xylazine (10 mg/kg) intramuscularly.

Ideally, the activation of the photosensitizer can be performed at themaximal absorption of 675 nm. In this example, the same laser used forraising the temperature of the tissue to cause release of the dye andagent from liposomes was used to activate the agent. A dye laser(Coherent, Palo Alto, Calif.), operated at 577 nm, was chosen to matchthe high absorption of blood at this wavelength thereby raising thetemperature of the iris vessel efficiently. The laser was coupled to aslit lamp biomicroscope as part of an ophthalmic laser delivery system.

The power of the laser was set according to calculations similar tothose used by other skilled practitioners for the power necessary towarm by 4 ° C. blood vessels embedded in a transparent medium. (See, forexample, Bebie et al. (1974) 52 Acta Ophthalmol. 13-36.) Thistemperature rise is sufficient to release the contents of theheat-sensitive liposomes. The power of the laser spot was 50 mW and itsdiameter was 400 mm.

Prior to the injection, the irides of both eyes were imaged to obtain abaseline. The liposome preparation was injected intravenously to yield adose of AlPcS₄ of 7.5 mg/kg. A portion of the iris, corresponding to 2clock hours, was treated. The number of pulses necessary to obtain aneffect was determined by pilot experiments. To control for the effect ofthe laser by itself, the left iris was exposed to the laser prior to theinjection of the photosensitive dye. The dye was then injected and theright iris was treated within 5 minutes of injection, although treatmentcan occur as early as practically feasible post-injection.

The heat-sensitive liposomes of the instant invention may release someof their content (10 to 15%) at body temperature. To assess the effectof this unencapsulated portion of the dose, a second control wasperformed. The same liposome preparation was lysed by heating it beyondthe phase-transition and 20% of the dose of the lysed preparation wasadministered to another rat. The iris was then treated within 5 minutesof the injection with the same laser parameters as above. The iris wascontinuously visualized for 15 minutes to detect any change in thevessels. To control for potential individual variability the treatmentwas followed 20 minutes later by an injection, to the same animal, ofintact liposomes and a different quadrant was treated with the samelaser delivery protocol.

The eyes were followed-up by obtaining red-free video images with a CCDCamera (Texas Instruments) coupled to one of the viewing arms of theslit lamp biomicroscope. The video output was recorded on magnetic tapewith a high frequency videorecorder (Sony, Tokyo, Japan) and laterdigitized with a frame grabber (Epix, Northbrook, Ill.).

Using 4 animals, its was established that 40 pulses of 0.5 sec yieldedvisible results. These parameters were thus adopted for the rest of thestudies.

When the right eye was occluded after liposome injections, vesselengorgement followed by vessel spasms, hemorrhages and local iris tissueexpansion were observed upon the delivery of 20 pulses. Local iristissue constriction then occurred at the end of 40 pulses. In contrast,in the left control eye treated prior to liposome injection, no effectwas noticed in the blood vessels or the iris tissue during the delivery.

The follow-up examinations with red-free imaging revealed, in thetreated eye, hemorrhages which cleared within a week, leaving the iristissue with non-perfused vessels. At 25 days, the occluded vessels inthe treated area could be seen as non-perfused vessels (ghost vessels).During the follow-up period which was done monthly for 8 months, noreperfusion of occluded vessels was observed. In contrast, the follow-upof the control eye did not reveal any ophthalmoscopically visiblepathology at any time point.

In the second experiment, following the injection of the photosensitizerat 20% of the encapsulated dose, no response was observed in the bloodvessels or the pupil when observed for fifteen minutes followingcompletion of 40 deliveries. When the intact liposome preparation wasinjected in the same animal and another quadrant was treated, theocclusive events mentioned above were observed after the first 20pulses.

The results demonstrate the success of laser-targeted occlusionperformed in accordance with the methods of the instant invention. Thecontrol experiments did not yield the effects which were observed whenthe photosensitive agent was released at high concentration from theliposomes and simultaneously irradiated. This assures that the occlusionwas not due to the direct effect of the laser on the tissue or to theactivation of the low dose of free photosensitizer. In contrast, thetreatment caused occlusion which lasted for the long term follow-up. Thefact that ghost vessels were visible from the seventh day onwardsprovides conclusive evidence that the observation of lack of perfusedvessels was due to occlusion and not to artifacts such as overlyingedematous fluid or exudate.

These results reveal that, photosensitizers can yield occlusive effectswhen activated during their presence inside the blood stream. Due to theshort passage of the agent, the activated photosensitive agent did nothave sufficient time to exit the lumen. This sheds some light on one ofthe mechanisms of photodynamic therapy as it indicates that thepenetration of the photosensitizer into the tissue or the cells is not aprerequisite for its effect.

But, most importantly, this study illustrates successful occlusion withthe instant invention's method of laser-targeted occlusion. The successin occluding vessels in the iris is significant because these aresubjected to a high pressure head. Moreover, abnormal new vessels suchas those encountered in macular degeneration are likely to be prone toeven more damage due to their defective endothelial lining.

EXAMPLE 6.2 Laser-Targeted Photo-Occlusion In Choroidal Vessels: Rodent

To demonstrate that the retina can be preserved while occluding thechoroid, experiments were performed in the choroid in pigmented rats.Liposomes with AlPcS₄ were injected, their content was released locallyby the laser pulse and the area was simultaneously irradiated toactivate the AlPcS₄. Fluorescein angiography was performed within twohours after the treatment, and revealed a slow filling and subsequentleakage of treated vessels in the choroid. These symptoms are indicativeof a fresh occlusive event. In contrast, the retinal capillaries wereclearly unaffected as illustrated by their perfusion and lack ofleakage. Camera motions in live video images provided depth resolutionwhich clearly indicated that the leakage was localized at the choroidallevel. The retinal capillaries overlying the treated area did notsustain damage as demonstrated by their similarity to those in theuntreated area. The animal was sacrificed 21/2 hours after the treatmentand histology was performed as described previously. The time wasselected to precede secondary damage to the RPE and photoreceptorslikely to occur within 2 to 3 days following ischemia in thechoriocapillaris. There was a dramatically reduced presence of HRP inthe choroid at the treatment site compared with heavy staining in thecontrol area. Most importantly, no damage was present in the RPE,photoreceptors and neuro-retina. These results clearly indicate thatlaser-targeted occlusion is not accompanied by the devastating damageobserved immediately after laser photocoagulation.

The clinical impact of a safe method to occlude CNV is significant Thesuccess in occluding normal vessels in the iris which are subjected to ahigh pressure head, indicates that laser-targeted photo-occlusion is asuccessful method of occlusion. Moreover, the success in occluding thenormal choroid without damage to the RPE, photoreceptors and outerneuro-retina, documented angiographically as well as histologically,clearly demonstrate that the method of the instant invention provides anapproach that can prevent the devastating injury induced by laserphotocoagulation.

EXAMPLE 6.3 Laser-Targeted Occlusion: Primates

A model of CNV can be created following protocols, each of which iswell-established and familiar to the skilled practitioner. See, forexample, Ryan (1982) 100 Arch. Ophthalmol. 1804-1809 and Miller et al.(1993) 111 Arch. Ophthalmol. 855-869. The presence of CNV will bedetermined by laser-targeted angiography. In eyes with two or morelesions with CNV, one lesion will be left as a control and the otherswill be subject to laser-targeted photoocclusion. The assignment will bemade by an independent observer.

The follow-up with red-free fundus images, laser-targeted angiography,and fluorescein angiography will be done weekly until the vessels in thecontrol areas begin their normal regression. The animals will then besacrificed and the eyes will be subject similarly to histology.

It is anticipated that laser-targeted occlusion will provide a safemethod to occlude CNV in primates and that essentially no damage to theRPE, photoreceptors and outer neuro-retina will be documented bothangiographically and histologically.

Laser-targeted occlusion as described herein can provide a therapy thatcan surpass laser photocoagulation and conventional photodynamic therapyfor a variety of reasons. For example, the results of the specificvisualization of the choroidal vasculature indicate that thetissue-reactive or photosensitive agent can be released in thesubretinal vasculature while avoiding release in the retinalcapillaries. Thus these vessels will not be damaged during theirradiation to activate the agent. Moreover, by irradiating immediatelyfollowing release from the liposomes, the damage can be limited to thevessels perfused. Accumulation of the agent in the interstitial tissuesand subsequent damage upon irradiation can be avoided. Additionally,CNVs are perfused by a slower flow than the normal choriocapillaris. Thetissue reactive or photosensitive agent can be released and the tissueirradiated only after enough time has elapsed to ensure clearance fromthe normal choriocapillaris. This ensures preservation of thechoriocapillaris which is crucial to the maintenance of the retinalpigment epithelium. Furthermore, nonthermal occlusion avoids extensivescarring and breaks in Bruch's membrane believed to increase therecurrence risk of neovascularization. Finally, laser targeted occlusionwill benefit a large portion of the population with macular degenerationby providing selective occlusion with better preservation of vision.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. For example,laser-targeted occlusion of vasculature can be practiced according tothe foregoing methods in body tissues other than the mammalian eye.Scope of the invention is thus indicated by the appended claims ratherthan by the foregoing description, and all changes which come within themeaning and range of equivalency of the claims are therefore intended tobe embraced therein.

What is claimed is:
 1. A method of occluding vasculature in a mammalianeye, comprising the steps of:(a) co-administering intravenously to amammal a fluorescent dye encapsulated within heat-sensitive liposomes, atissue-reactive agent activated by irradiation and a tissue-specificfactor, said tissue-specific factor effective to impair growth orregeneration of a blood vessel or blood sinus; (b) selectively heatingvasculature at a pre-determined anatomical locus within the eye suchthat the heat-sensitive liposomes present at said locus release locallytheir contents wherein said tissue-reactive agent remains inactive; (c)monitoring clearance of said fluorescent dye from vasculature at saidlocus with normal blood flow, while simultaneously monitoring andvisualizing persistence of said fluorescent dye within vasculature atsaid locus having subnormal blood flow; and, after having visualizedpersistent fluorescence in said vasculature having subnormal blood flow,(d) activating the tissue-reactive agent present in said vasculaturehaving subnormal blood flow, such that the activated agent chemicallyoccludes said vasculature.
 2. The method of claim 1 wherein saidtissue-specific factor comprises a factor effective to cause regressionof a blood vessel or blood sinus.
 3. The method of claim 1 wherein saidtissue-specific factor comprises a factor which binds to endothelialcell receptors of a growing or regenerating blood vessel or blood sinus.4. The method of claim 1 wherein said tissue-specific factor effectiveto impair growth or regeneration is an antibody.
 5. The method of claim4 wherein said antibody is an antibody against vascular endothelialgrowth factor.
 6. The method of claim 4 wherein said antibody is anantibody against fibroblast growth factor.
 7. The method of claim 1wherein step (a) further comprises co-administering an anti-inflammatoryagent encapsulated within heat-sensitive liposomes.
 8. The method ofclaim 1 wherein step (a) further comprises co-administering anantibiotic encapsulated within heat-sensitive liposomes.
 9. The methodof claim 8 wherein the antibiotic is an anti-bacterial, anti-fungal,anti-neoplastic or anti-parasitic antibiotic.
 10. A method of chemicallyoccluding a blood vessel or blood sinus in a mammalian eye, comprisingthe steps of:(a) co-administering intravenously to a mammal afluorescent dye and a tissue-specific factor, said dye and said factorbeing encapsulated within heat-sensitive liposomes, said factoreffective to impair growth or regeneration of vasculature; (b)non-invasively heating tissue at a pre-determined anatomical locuswithin the eye of said mammal comprising a blood vessel or sinus,thereby to lyse said heat-sensitive liposomes containing saidfluorescent dye and to release the contents thereof at said locus; (c)exciting said fluorescent dye; (d) monitoring clearance of saidfluorescent dye from a blood vessel or sinus at said locus with normalblood flow, while simultaneously monitoring and visualizing persistenceof said fluorescent dye within a blood vessel or sinus at said locushaving subnormal blood flow; and, after having visualized persistentfluorescence in said blood vessel or sinus blood flow, (e) exposing saidblood vessel or sinus at said locus to said tissue-specific factordisposed within said blood vessel or sinus, thereby to impair growth orregeneration sufficient to occlude said blood vessel or sinus.
 11. Themethod of claim 10 wherein said tissue-specific factor comprises afactor effective to cause regression of a blood vessel or blood sinus.12. The method of claim 10 wherein said tissue-specific factor comprisesa factor which binds to endothelial cell receptors of a growing orregenerating blood vessel or blood sinus.
 13. The method of claim 10wherein said tissue-specific factor effective to impair growth orregeneration is an antibody.
 14. The method of claim 13 wherein saidantibody is an antibody against vascular endothelial growth factor. 15.The method of claim 13 wherein said antibody is an antibody againstfibroblast growth factor.
 16. The method of claim 10 wherein step (a)further comprises co-administering an anti-inflammatory agentencapsulated within heat-sensitive liposomes.
 17. The method of claim 10wherein step (a) further comprises co-administering an antibioticencapsulated within heat-sensitive liposomes.
 18. The method of claim 17wherein the antibiotic is an anti-bacterial, anti-fungal,anti-neoplastic or anti-parasitic antibiotic.
 19. A method of occludingvasculature in a mammalian eye comprising the steps of:(a) administeringheat-sensitive liposomes intravenously to said mammal, said liposomeshaving a fluorescent dye encapsulated therein; (b) irradiating apre-determined anatomical locus within the eye with a first laser beam,said first beam selectively and non-invasively heating the vasculatureat said pre-determined anatomical locus such that the heat-sensitiveliposomes accumulated at said locus are lysed and said fluorescent dyeis released into the vasculature at said locus; (c) identifying a bloodflow origin within said vasculature by visualizing an advancingblood/dye boundary within a feeder blood vessel that supplies blood to avascular abnormality; and, (d) occluding said feeder blood vessel with asecond laser beam focused on said blood/dye boundary.
 20. The method ofclaim 19 wherein said second beam is effective to cause heat damage tosaid feeder blood vessel such that said feeder blood vessel iscoagulated and thereby occluded.
 21. The method of claim 19 wherein saidsecond beam is effective to activate said fluorescent dye such thattissue-reactive by-products are produced therefrom and said feeder bloodvessel is thereby occluded.
 22. A method of chemically occludingabnormal vasculature in a mammalian eye comprising the steps of:(a)administering heat-sensitive liposomes intravenously to said mammal,said liposomes having a tissue-reactive agent and a fluorescent dyeencapsulated therein; (b) irradiating a pre-determined anatomical locuswithin the eye with a first laser beam, said first beam selectively andnon-invasively heating vasculature at said pre-determined anatomicallocus such that the heat-sensitive liposomes accumulated at said locusare lysed and said tissue-reactive fluorescent dye is released into thevasculature at said locus; (c) monitoring clearance of said fluorescentdye from vasculature at said locus with normal blood flow whilesimultaneously monitoring and visualizing persistence of said dye withinvasculature at said locus with subnormal blood flow; and after havingvisualized persistent fluorescence in said vasculature having subnormalblood flow, (d) irradiating with a second laser beam said vasculaturewith subnormal blood flow by focusing said second laser beam onvasculature containing persistent fluorescent dye, said second beamactivating the tissue-reactive fluorescent dye persisting in saidvasculature such that said vasculature are chemically damaged andthereby occluded.
 23. The method of claim 22 wherein said fluorescentdye is said tissue-reactive agent.